1. Field of the Invention
The invention generally relates to agents for the treatment of sickle-cell disease. In particular, the invention provides 5-membered heterocyclic anti-sickling agents that are highly effective and non-toxic, and methods for their use.
2. Background of the Invention
Sickle cell disease is one of the most prevalent hematologic genetic disorders in the world (Ingram, 1956; Pauling, et al. 1949) that occurs as a result of a single point mutation of Glu6 in Hb to Val6 in sickle hemoglobin (HbS). Two quaternary structures are known for Hb, the deoxy conformation (tense), and the oxygenated conformation (relaxed). When the allosteric equilibrium is shifted toward the relaxed state, a high-affinity Hb is obtained that readily binds and holds oxygen, while the converse is true for the tense state. Perutz (1970) and Baldwin & Chothia (1979) elucidated at atomic resolution the tetrameric structures of the tense (T) and relaxed (R) forms of Hb. The tetramer is composed of two αβ dimers that are arranged around a twofold axis of symmetry. This arrangement yields a central water cavity, with two openings; the α- and β-clefts. The source of the tension in the T state is due to crosslinking salt bridges and hydrogen bonds between the subunits, as well as preferential binding of an indigenous allosteric effector of Hb, 2,3-diphosphoglycerate (2,3-DPG) that stabilize the T state by forming salt bridges between the two β-subunits (Arnone, 1992). The T-R transition occurs as a result of uptake of oxygen which leads to the disruption of many of the T state intersubunit interactions, as well as expulsion of the 2,3-DPG. The allosteric transition results in a rotation of the α1β1 dimer relative to the α2β2 dimer by 12–15° (Baldwin & Chothia, 1979). The R state structure has a smaller central water cavity, as well as fewer intersubunit salt bridges and hydrogen bonds. For a long period of time, the allosteric equilibrium of Hb embodied in the two-state MWC model (Monod, et. al., 1965) was believed to involve only the T-R transition, and the R state quaternary structure was thought to be the only relaxed conformer. However, recent crystallographic and other studies have revealed the existence of multi relaxed Hb states, including R2 and others that exist in solution with R (Silva, et al. 1992; Smith, et al., 1991; Mueser, et al., 2000). There is still a controversy about the physiological importance of all these relaxed states, and how they relate to one another in Hb allostery. Silva et al., (1992) and Smith et al., (1991) suggested that the R2 quaternary structure is an intermediate between the T and R structures. Further analysis has shown that R2 is not an intermediate in the T to R transition, but rather, it is another relaxed end-state structure (Janin & Wodak, 1993; Doyle, et al., 1992). Srinivasan & Rose (1994) have further suggested that R2 may be the physiologically relevant end state and that the R structure is an intermediate structure trapped between the R2 and T states by the high-salt crystallization conditions. In contrast, the R2 structure formation is believed to be favored by low-salt that mimic the in vivo condition (Silva et al., 1992; Srinivasan & Rose, 1994).
Hb and HbS have almost identical positions for all amino acids, even in the A helix of the chains where the mutation occurs. The presence of the Val6 results in hydrophobic interaction between the mutation region of one Hb molecule and a region defined by Phe85 and Leu88 in the heme pocket of another Hb molecule. This interaction occurs only in the deoxygenated HbS (deoxyHbS), and induces polymerization of the deoxyHbS molecules into fibers. The formation of HbS polymers causes the normally flexible red blood cells to adopt rigid, sickle like shapes that block small capillaries and cause both local tissue damage and severe pain. The disease is also characterized by other symptoms, including hemolysis, which gives rise to anemia and jaundice, elevation of bilirubin level leading to high incidence of gall stones and impairment of hepatic excretory function. Other clinical features include leg ulceration, pneumonia, enlarged liver and spleen. Other studies on the gellation of deoxyHbS and various Hb variants have also provided crucial information on other contact points on the Hb that are important in stabilizing the HbS fibre (Adachi & Asakura, 1980; Bunn, et al., 1986). There are various therapeutic strategies to treat sickle cell disease (SCD), including (1) Pharmacological modulation of fetal hemoglobin (HbF): HbF has been shown to decrease HbS polymerization, and there are several agents that are known to induce HbF formation by possibly reactivating the genetic switch for HbF (Olivieri & Weatherall, 1998). Examples of such agents include 5-azacytidine, hydroxyurea and cytosine arabinoside (Mehanna, 2001). Unfortunately, there are serious toxic side effects associated with this therapy as a result of high doses and frequency of administration (Edelstein, 1985), (2) Bone marrow transplantation: Bone marrow transplant has also been used as a total gene replacement therapy for HbS in extreme cases (Hillery, 1998, Johnson, 1985). This approach is very expensive and has its own inherent toxicities and risks (Hillery, 1998), (3) Blood transfusion: This is one of the most common SCD therapies, however, repeated blood transfusions are known to be associated with the risks of infectious diseases, iron overload and allergic reactions (Ballas, 1999), (4) Opioid analgesics: This therapy is necessary to deal with pain crisis, however, opioid therapy often results in addiction and/or seizures and/or depression, (Ballas, 1999), (5) Erythrocyte membrane acting agents: Since the sickling process is partly dependent on intracellular concentration of sickle Hb, agents that induce cell swelling (Asakura, 1980) or inhibit cell dehydration (Orringer & Berkwitz, 1986) could decrease the HbS concentration and help delay the polymerization process, and (6) Antigelling agent or HbS modifiers: These compounds interfere with the mechanism of polymerization by either binding directly to or near contact site(s) of the deoxyHbS to inhibit the polymerization process or act directly on HbS to shift the allosteric equilibrium to the more soluble high-affinity HbS.
In blood, Hb is in equilibrium between the T and the relaxed states. The Hb delivers oxygen via an allosteric mechanism, and the ability for the Hb to release or take oxygen can be regulated by allosteric effectors. The allosteric equilibrium between the T and relaxed states (FIG. 1) shows a typical oxygen equilibrium curve (OEC) for Hb, i.e. a plot of the percentage of oxygen bound by Hb against the partial pressure of oxygen. When the allosteric equilibrium is shifted towards the relaxed state (left shift of the curve), a high-affinity Hb is obtained that more readily binds and holds oxygen while a shift toward the T state (right shift of the curve) results in a low-affinity Hb that more easily releases oxygen. An increase in the naturally occurring allosteric effector, 2,3-DPG in red cells right shifts the OEC as does an increase in temperature and decrease in pH (Reeves, 1980). An increase in pH and lowering of the temperature and DPG levels left shifts the OEC. The degree of shift in the OEC is reported as an increase or decrease in P50 (partial pressure of oxygen at 50% Hb saturation). Regulating the allosteric equilibrium to the relaxed conformation has been of been of interest in medicine. In particular, the identification of non-toxic compounds that efficiently bind to HbS and produce high-affinity HbS which does not polymerize have been clinically evaluated as antisickling agents to treat SCD. There is an ongoing need to identify such compounds to be used as antisickling agents to treat sickle cell anemia. See, for example, the use of vanillin (Abraham, 1991), 12C79 (Fitzharris, 1985), furfural (Zaugg, et al., 1997), and substituted isothiocyanates (Park, et al. 2003).